Methods and compositions for treatment of tumors using nucleic acid ligands to platelet-derived growth factor

ABSTRACT

A method is provided for treating solid tumors comprising administering a composition comprising a PDGF aptamer and a cytotoxic agent. In a preferred embodiment the PDGF aptamer is identified using the SELEX process for the Systematic Evolution of Ligands by Exponential enrichment. A method is also provided for reducing the interstitial fluid pressure (IFP) of a solid tumor comprising administering a PDGF aptamer. Finally, a method is provided for increasing the uptake of cytotoxic agents into a tumor comprising administering a composition comprising a PDGF aptamer and a cytotoxic agent.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/791,367, filed Mar. 2, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/859,724, filed May 17, 2001, now U.S. Pat. No.6,699,843, which claims the benefit of U.S. Provisional Application Ser.No. 60/205,006, filed May 17, 2000. U.S. patent application Ser. No.09/859,724 is a continuation in part of U.S. patent application Ser. No.08/479,725, filed Jun. 7, 1995, now U.S. Pat. No. 5,674,685, and also acontinuation in part of U.S. patent application Ser. No. 08/479,783,filed Jun. 7, 1995, now U.S. Pat. No. 5,668,264, and also a continuationin part of U.S. patent application Ser. No. 08/618,693, filed Mar. 20,1996, now U.S. Pat. No. 5,723,594, each entitled “High Affinity PDGFNucleic Acid Ligands.” U.S. patent application Ser. No. 09/859,724 isalso a continuation in part of U.S. patent application Ser. No.08/991,743, filed Dec. 16, 1997, now U.S. Pat. No. 6,229,002, entitled“Platelet Derived Growth Factor (PDGF) Nucleic Acid Ligand Complexes.”Each of the foregoing applications is incorporated herein by referencein their entirety.

FIELD OF THE INVENTION

This invention relates generally to a method for increasing the uptakeof drugs into tumors by treatment with a nucleic acid ligand to PDGF incombination with a cytotoxic agent. The method used for identifyingnucleic acid ligands to PDGF is called SELEX, an acronym for SystematicEvolution of Ligands by Exponential enrichment. The method of thepresent invention is useful for increasing the therapeutic effectivenessof cytotoxic agents.

BACKGROUND OF THE INVENTION

The Systematic Evolution of Ligands by Exponential Enrichment (SELEX)process is a method for the in vitro evolution of nucleic acid moleculeswith highly specific binding to target molecules and is described inU.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990,entitled “Systematic Evolution of Ligands by EXponential Enrichment,”now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands,”and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled “Methodsfor Identifying Nucleic Acid Ligands,” each of which is specificallyincorporated herein by reference in its entirety. Each of theseapplications, collectively referred to herein as the SELEX PatentApplications, describes a fundamentally novel method for making anucleic acid ligand to any desired target molecule.

The SELEX process provides a class of products which are referred to asnucleic acid ligands or aptamers, each having a unique sequence, andwhich has the property of binding specifically to a desired targetcompound or molecule. Each SELEX-identified nucleic acid ligand is aspecific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets. The SELEX method applied tothe application of high affinity binding involves selection from amixture of candidate oligonucleotides and step-wise iterations ofbinding, partitioning and amplification, using the same generalselection scheme, to achieve virtually any desired criterion of bindingaffinity and selectivity. Starting from a mixture of nucleic acids,preferably comprising a segment of randomized sequence, the SELEX methodincludes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

It has been recognized by the present inventors that the SELEX methoddemonstrates that nucleic acids as chemical compounds can form a widearray of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, bothentitled “Method for Selecting Nucleic Acids on the Basis of Structure,”describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177 and U.S.Pat. No. 6,011,577, both entitled “Systematic Evolution of Ligands byExponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX,” describe a SELEX based method for selecting nucleicacid ligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine,” describes a method foridentifying highly specific nucleic acid ligands able to discriminatebetween closely related molecules, which can be non-peptidic, termedCounter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolutionof Ligands by EXponential Enrichment: Solution SELEX,” describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” describes oligonucleotides containingvarious 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolutionof Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Blended SELEX,” respectively. These applications allow thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

The SELEX method further encompasses combining selected nucleic acidligands with lipophilic compounds or non-immunogenic, high molecularweight compounds in a diagnostic or therapeutic complex as described inU.S. Pat. No. 6,011,020, entitled “Nucleic Acid Ligand Complexes.” Eachof the above described patent applications which describe modificationsof the basic SELEX procedure are specifically incorporated by referenceherein in their entirety.

One approach to increasing the effectiveness of existing anti-cancerdrugs for the treatment of solid malignancies is to augment the uptakeof the drugs into tumors, and thereby obtain increased therapeuticconcentration without elevating the adverse side-effects. Most solidtumors display an increased interstitial fluid pressure (IFP). Themolecular mechanisms causing increased tumor IFP are poorly understood.However, tumor stroma involvement in IFP control has been demonstrated.(Gullino et al. (1964) Cancer Res. 24:780-797; Philips et al. (1990) J.Natl Cancer Inst. 82:1457-1469; Jain (1987) Cancer Res. 47:3039-3051).It has been suggested that high tumor IFP prevents drug transport fromthe circulation into the tumor interstitium. The reduction of tumor IFPtherefore is a target for efforts to increase tumor drug uptake. (Jain(1996) Science 271:1079-1080). Several agents which induce a lowering ofIFP, and thereby increase the transcapillary transport in experimentalmurine tumors have been identified, including nicotinamide (Lee et al.(1992) Cancer Res. 52:3237-3240), TNF-α (Kristensen et al. (1996) Br. J.Cancer 74:533-536) and dexamethasone (Kristjansen et al. (1993) CancerRes. 53:4764-4766).

Platelet-derived growth factor (PDGF) and the cognate tyrosine kinasereceptors are potent mitogens for mesenchymal cells. In addition to itsgrowth promoting effects, PDGF-BB is involved also in the regulation ofIFP. After dextran-induced anaphylaxis and lowering of IFP in rat skin,local administration of PDGF-BB results in normalized IFP. PDGFreceptors are expressed in the tumor stroma of many common solid tumors,e.g. lung, colon and breast carcinomas. Based on these observations theeffects of PDGF antagonists on tumor IFP, tumor transcapillary transportand therapeutic effects of cytotoxic drugs were investigated.

SUMMARY OF THE INVENTION

The present invention includes a method for treating tumors, morespecifically, solid tumors comprising administering to a host atherapeutically effective dose of a composition comprising a PDGFaptamer and a cytotoxic agent. In a preferred embodiment the PDGFaptamer is identified using the SELEX process for the SystematicEvolution of Ligands by EXponential enrichment. The present inventionalso includes a method for reducing the interstitial fluid pressure(IFP) of a tumor, more specifically, a solid tumor comprisingadministering a PDGF nucleic acid ligand. Finally, the present inventionincludes a method for increasing the uptake of cytotoxic agents into atumor comprising administering to a host a composition comprising a PDGFaptamer and a cytotoxic agent. The present invention provides a novelmethod to increase drug uptake and therefore the therapeuticeffectiveness of chemotherapy, by treatment with a PDGF inhibitor inconjunction with the therapeutic agent. As illustrated below, treatmentwith a PDGF aptamer decreases interstitial hypertension in these tumorsthereby increasing the uptake of the therapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B show the molecular description of the PDGF aptamer usedin the present study ((SEQ ID NO:1), FIG. 1A) and the aptamer that wasused as the control. ((SEQ ID NO:2), FIG. 1B).

FIG. 2 illustrates graphically the reduction in tumor interstitial fluidpressure (IFP) upon treatment of PROb tumors with a PDGF specificaptamer. Average tumor IFP in the animals treated with asequence-scrambled control oligonucleotide (n=5) and PDGF-B specificaptamer (n=7) is depicted. Bars represent S.E.M; * p<0.05

FIG. 3 depicts the distribution of PDGF β-receptors and PDGF-AB/BB inPROb tumors. Morphological analysis of sections from PROb tumor cellsrevealed three discrete zones in the investigated tumors (FIG. 3A).Towards the exterior a cell rich zone containing proliferative cellsarranged in glandular structures was evident. A zone further towards thecentral part of the tumors contained apoptotic cells and cell debris,whereas the central parts were largely acellular, but containedextracellular matrix deposits. Inflammatory infiltrates were not evidentin the investigated tumors. Immunohistochemical analyses showed thatPDGF-AB/BB was expressed in blood vessels (FIG. 3B) and in the centralparts of tumors by cells morphologically identified as macrophages (FIG.3C). A weak but clearly visible staining of PDGF β-receptors was presentin stromal structures in large parts of the tumors, especially aroundtumor glands (FIG. 3D). PDGF β-receptor staining was completely blockedby addition of a peptide corresponding to the amino acids 981-994 of thehuman/murine PDGF β-receptor at a 10-fold molar excess to the anti-PDGFβ-receptor antibody (FIG. 3E).

FIG. 4 illustrates graphically the reduction of IFP upon treatment ofSCID-Mice bearing subcutaneous KAT-4 tumors (human anaplastic thyroidcarcinoma with PDGF aptamers.

FIG. 5 illustrates graphically the increased uptake of thechemotherapeutic agent Taxol upon pretreatment of KAT-4 tumors with PDGFaptamers.

DETAILED DESCRIPTION OF THE INVENTION

Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

As used herein a “nucleic acid ligand” is a non-naturally occurringnucleic acid having a desirable action on a target. Nucleic acid ligandsare often referred to as “aptamers.” A desirable action includes, but isnot limited to, binding of the target, catalytically changing thetarget, reacting with the target in a way which modifies/alters thetarget or the functional activity of the target, covalently attaching tothe target as in a suicide inhibitor, facilitating the reaction betweenthe target and another molecule. In a preferred embodiment, the actionis specific binding affinity for a target molecule, such target moleculebeing a three dimensional chemical structure other than a polynucleotidethat binds to the nucleic acid ligand through a mechanism whichpredominantly depends on Watson/Crick base pairing or triple helixbinding, wherein the nucleic acid ligand does not have the knownphysiological function of being bound by the target molecule. In thepresent invention, the target is PDGF or regions thereof. Nucleic acidligands include nucleic acids that are identified from a candidatemixture of nucleic acids, said nucleic acid ligand being a ligand of agiven target, by the method comprising: a) contacting the candidatemixture with the target, wherein nucleic acids having an increasedaffinity to the target relative to the candidate mixture may bepartitioned from the remainder of the candidate mixture; b) partitioningthe increased affinity nucleic acids from the remainder of the candidatemixture; and c) amplifying the increased affinity nucleic acids to yielda ligand-enriched mixture of nucleic acids.

As used herein a “candidate mixture” is a mixture of nucleic acids ofdiffering sequence from which to select a desired ligand. The source ofa candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

As used herein, “nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded, and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

“SELEX” methodology involves the combination of selection of nucleicacid ligands that interact with a target in a desirable manner, forexample binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology was employed to obtain nucleic acidligands to PDGF. See U.S. Pat. No. 5,674,685, issued Oct. 7, 1997, U.S.Pat. No. 5,668,264, issued Sep. 16, 1997 and U.S. Pat. No. 5,723,594,issued Mar. 3, 1998, each entitled “High Affinity PDGF Nucleic AcidLigands,” and U.S. Pat. No. 6,229,002, issued, May 8, 2001, entitled“Platelet Derived Growth Factor (PDGF) Nucleic Acid Ligand Complexes.”

The SELEX methodology is described in the SELEX Patent Applications.

“SELEX target” or “target” means any compound or molecule of interestfor which a ligand is desired. A target can be a protein, peptide,carbohydrate, polysaccharide, glycoprotein, hormone, receptor, antigen,antibody, virus, substrate, metabolite, transition state analog,cofactor, inhibitor, drug, dye, nutrient, growth factor, etc. withoutlimitation. In this application, the SELEX target was PDGF.

A “cytotoxic agent” is any substance used to destroy tumor cells. Themethod of this invention can be used with any systemically administratedcytotoxic agent including, but not limited to, Bleomycin, Cisplatin, andPt analogues; Carboplatin and Iproplatin, Cyclophosphamide,Daunorubicin, Doxofluoridine, Doxorubicin, Etoposide, Epirubicin,5-Flurouracil, Gemzar, Ifosfamide, Melphalan, Methotrexate, Mithramycin,Mitomycin C, Mitoxanthrone, Streptozotocin, Taxol and Taxotere,Vincristine, Vinblastine, Vindesine, Vinorelbine, Topotecan and CPT-11.

“Therapeutic” as used herein, includes treatment and/or prophylaxis.When used, therapeutic refers to humans, as well as, other animals.

“Pharmaceutically or therapeutically effective dose or amount” refers toa dosage level sufficient to induce a desired biological result. Thatresult may be the delivery of a pharmaceutical agent, alleviation of thesigns, symptoms or causes of a disease or any other desirous alterationof a biological system.

A “host” is a living subject, human or animal, into which a drug orcytotoxic agent is administered.

Note, that throughout this application various citations are provided.Each citation is specifically incorporated herein in its entirety byreference.

The present invention includes a method for treating solid tumorscomprising administering to a host a therapeutically effective dose of acomposition comprising a PDGF aptamer and a cytotoxic agent. In apreferred embodiment the PDGF aptamer is identified using the SELEXmethodology. The SELEX process is described in U.S. patent applicationSer. No. 07/536,428, entitled “Systematic Evolution of Ligands byExponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096,entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163 (see alsoWO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands.”These applications, each specifically incorporated herein by reference,are collectively called the SELEX Patent Applications.

The SELEX process provides a class of products that are nucleic acidmolecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

In its most basic form, the SELEX process may be defined by thefollowing series of steps.

1. A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below; (b) to mimic a sequenceknown to bind to the target; or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

2. The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

3. The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

4. Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5. By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer unique sequences, andthe average degree of affinity of the nucleic acids to the target willgenerally increase. Taken to its extreme, the SELEX process will yield acandidate mixture containing one or a small number of unique nucleicacids representing those nucleic acids from the original candidatemixture having the highest affinity to the target molecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. patent application Ser. No. 07/960,093,filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, bothentitled “Method for Selecting Nucleic Acids on the Basis of Structure,”describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177 and U.S.Pat. No. 6,001,577, both entitled “Systematic Evolution of Ligands byExponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX,” all describe a SELEX based method for selecting nucleicacid ligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine,” describes a method foridentifying highly specific nucleic acid ligands able to discriminatebetween closely related molecules, termed Counter-SELEX. U.S. Pat. No.5,567,588, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Solution SELEX,” describes a SELEX-based method whichachieves highly efficient partitioning between oligonucleotides havinghigh and low affinity for a target molecule. U.S. Pat. No. 5,496,938,entitled “Nucleic Acid Ligands to HUV-RT and HIV-1 Rev,” describesmethods for obtaining improved nucleic acid ligands after SELEX has beenperformed. U.S. Pat. No. 5,705,337, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Chemi-SELEX,” describes methods forcovalently linking a ligand to its target.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” describes oligonucleotides containingvarious 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Chimeric SELEX,” and U.S. Pat. No.5,683,867, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Blended SELEX,” respectively. These applications allow thecombination of the broad array of shapes and other properties, and theefficient amplification and replication properties, of oligonucleotideswith the desirable properties of other molecules.

In U.S. Pat. No. 5,496,938, methods are described for obtaining improvednucleic acid ligands after the SELEX process has been performed. Thispatent, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” isspecifically incorporated herein by reference.

One potential problem encountered in the diagnostic use of nucleic acidsis that oligonucleotides in their phosphodiester form may be quicklydegraded in body fluids by intracellular and extracellular enzymes, suchas endonucleases and exonucleases, before the desired effect ismanifest. Certain chemical modifications of the nucleic acid ligand canbe made to increase the in vivo stability of the nucleic acid ligand orto enhance or to mediate the delivery of the nucleic acid ligand. See,e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993,now abandoned and U.S. Pat. No. 5,660,985, both entitled “High AffinityNucleic Acid Ligands Containing Modified Nucleotides,” and U.S. patentapplication Ser. No. 09/362,578, filed Jul. 28, 1999, entitled“Transcription-free SELEX,” each of which is specifically incorporatedherein by reference in its entirety. Modifications of the nucleic acidligands contemplated in this invention include, but are not limited to,those which provide other chemical groups that incorporate additionalcharge, polarizability, hydrophobicity, hydrogen bonding, electrostaticinteraction, and fluxionality to the nucleic acid ligand bases or to thenucleic acid ligand as a whole. Such modifications include, but are notlimited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.In preferred embodiments of the instant invention, the nucleic acidligands are RNA molecules that are 2′-fluoro (2′-F) modified on thesugar moiety of pyrimidine residues.

The modifications can be pre- or post-SELEX process modifications.Pre-SELEX process modifications yield nucleic acid ligands with bothspecificity for their SELEX target and improved in vivo stability.Post-SELEX process modifications made to 2′-OH nucleic acid ligands canresult in improved in vivo stability without adversely affecting thebinding capacity of the nucleic acid ligand.

Other modifications are known to one of ordinary skill in the art. Suchmodifications may be made post-SELEX process (modification of previouslyidentified unmodified ligands) or by incorporation into the SELEXprocess.

The nucleic acid ligands to PDGF of the invention are prepared throughthe SELEX methodology that is outlined above and thoroughly enabled inthe SELEX applications incorporated herein by reference in theirentirety.

As noted above, the cytotoxic agent can be any substance used in theprevention, diagnosis, alleviation, treatment or cure of disease. Morespecifically, the cytotoxic agent can be selected from any systemicallyadministrated agent including, but not limited to, Bleomycin, Cisplatin,and Pt analogues; Carboplatin and Iproplatin, Cyclophosphamide,Daunorubicin, Doxofluoridine, Doxorubicin, Etoposide, Epirubicin,5-Flurouracil, Gemzar, Ifosfamide, Melphalan, Methotrexate, Mithramycin,Mitomycin C, Mitoxanthrone, Streptozotocin, Taxol and Taxotere,Vincristine, Vinblastine, Vindesine, Vinorelbine, Topotecan and CPT-11.

Various delivery systems are known in the art and can be used toadminister the therapeutic composition comprising the PDGF aptamer andcytotoxic agent of the invention, e.g., aqueous solution, encapsulationin liposomes, microparticles, and microcapsules.

Therapeutic compositions of the invention may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and the nucleic acid ligand constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

Once the therapeutic composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready to use form or requiring reconstitution immediately prior toadministration. The manner of administering formulations containing thecompositions for systemic delivery may be via subcutaneous,intramuscular, intravenous, intranasal or vaginal or rectal suppository.

The amount of the composition which will be effective in the treatmentof a particular disorder or condition will depend on the nature of thedisorder of condition, which can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness or advancement of the disease orcondition, and should be decided according to the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curved derived from in vitro or animal model test systems.For example, an effective amount of the composition of the invention isreadily determined by administering graded doses of the composition ofthe invention and observing the desired effect.

The following examples are provided to explain and illustrate thepresent invention and are not intended to be limiting of the invention.

The PDGF-B aptamer used in the present study ((SEQ ID NO:1), FIG. 1A)was produced by the SELEX method. (Tuerk and Gold (1990) Science249:505-510, which is incorporated herein by reference in its entirety).The modified DNA aptamer, linked to 40 kDa polyethylene glycol, has ahigh affinity for PDGF-B with a Kd of ˜0.1 nM. (Green et al. (1996)Biochemistry 35:14413-14424; Floege et al. (1999) Am. J. Pathol.154:169-179; U.S. Pat. No. 6,229,002, issued May 8, 2001, ((SEQ IDNO:146), FIG. 9A), each of which is incorporated herein by reference inits entirety). A sequence-scrambled analog of the PDGF-B aptamer wasused as a control. ((SEQ ID NO:2), FIG. 1B). This oligonucleotide has aKd for PDGF-BB in the micromolar range. (Floege et al. (1999) Am. J.Pathol. 154:169-179; U.S. Pat. No. 6,229,002, issued May 8, 2001, ((SEQID NO:147), FIG. 9B)).

Example 1 describes the method used to treat PROb tumor-bearing ratswith the PDGF-B specific aptamer. As can be seen in FIG. 2 treatment ofPROb tumor-bearing rats with the PDGF-B specific aptamer resulted in adecrease in tumor IFP when compared to rats treated with the controlaptamer. The mean IFP in control aptamer-treated tumors was 14.6±1.2 mmHg (±S.E.M.) and 9.7±1.6 mm Hg (±S.E.M.) in tumors treated with thePDGF-B specific aptamer. The method used to determine IFP is describedin Example 2.

PROb tumors were analyzed with regard to morphology, as well asdistribution of PDGF-AB/BB and PDGF α-receptors as described in Example3. The tumors displayed a heterogeneous morphology. At the tumorperiphery, tumor cells were arranged in glandular structures, whereasmore centrally, tumor cells were less abundant and less well organized(FIG. 3A). The central part was basically acellular (FIG. 3A).Expression of PDGF-AB/BB in PROb tumors was found in blood vessels andpossibly in extravascular stromal cells surrounding tumor glands (FIG.3B). In the central part of the tumors, few if any tumor cells werepresent, but strongly PDGF-AB/BB positive cells were seen (FIG. 3C). Inno part of the tumors could PDGF-AB/BB positive tumor cells be clearlydiscerned. PDGF β-receptors were found in vascular cells of largervessels, and in unidentified, possibly microvascular, cells in thestroma (FIG. 3D). The absence of PDGF-AB/BB and P-receptor expression bycarcinoma cells in PROb tumors, is in agreement with the characteristicsof cultured PROb cells (data not shown).

Prompted by these findings, the effects of treatment with PDGF aptamer(SEQ ID NO:1) were tested on the KAT-4 tumor model (Examples 4 and 5).Both of these tumor models showed PDGF receptor expression in tumorstroma but not on tumor cells. As can be seen in FIGS. 4 and 5 treatmentwith PDGF aptamers lowers IFP in KAT-4 tumors and increases the uptakeof Taxol.

EXAMPLES

Materials and Methods

Tissue culture. Cells were cultured under standard conditions and alltissue culture media were supplemented with 10% fetal bovine serum (FBS)and antibiotics, unless otherwise stated. PAE cells were maintained inF12 culture medium (Sigma). PROb cells were kept in Dulbecco's ModifiedEagle's Medium (Sigma).

PDGF/PDGF receptor inhibitors. The PDGF-B aptamer used in the presentstudy was produced by the Systematic Evolution of Ligands by ExponentialEnrichment (SELEX) method. (Tuerk and Gold (1990) Science 249:505-510,which is incorporated herein by reference in its entirety). The modifiedDNA aptamer, linked to 40 kDa polyethylene glycol, has a high affinityfor PDGF-B with a Kd of ˜0.1 nM. (Green et al. (1996) Biochemistry35:14413-14424; Floege et al. (1999) Am. J. Pathol. 154:169-179, each ofwhich is incorporated herein by reference in its entirety). The aptamershows biphasic clearance in rats following iv injection, approximately47% is cleared with a half-life of 32 minutes, while the remainder iscleared with a half-life of 135 minutes. (Floege et al. (1999) Am. J.Pathol. 154:169-179). As a control, a sequence-scrambled analog of thePDGF-B aptamer was used. This oligonucleotide has a Kd for PDGF-BB inthe micromolar range. (Floege et al. (1999) Am. J. Pathol. 154:169-179).

Example 1 Tumor Establishment and Treatment of PROb Tumors with PDGFInhibitors

Subcutaneously growing PROb tumors (Martin et al. (1996) Int. J. Cancer65:796-804) were established in BDIX rats by injection of 5×10⁶ tumorcells in 50 μL of PBS in the flank. The rats were kept underpathogen-free conditions and were fed ad libitum. They were monitoredregularly for tumor growth and experiments were performed 8-12 weeksafter tumor cell implantation on rats bearing tumors ranging in sizebetween 0.6 cm³ and 7.6 cm³. The PDGF-B specific aptamer, and a controlaptamer (see above), were given as i.p. injections in 2 mL PBS twicedaily for 4 consecutive days at a dose of 7 mg×kg⁻¹×day⁻¹. All animalexperiments described in the present report were approved by the EthicalCommittee for Animal Experiments in Uppsala, Sweden.

Example 2 Measurement of Tumor IFP

Tumor IFP was measured using the wick-in-needle technique (Wiig et al.(1982) Scan. J. Clin. Lab. Invest. 42:159-164). Briefly, rats wereanaesthetized using isofluran in a mixture of O₂ and air. A standard23-gauge needle filled with nylon-floss and saline, supplemented with 50IE/mL of heparin was inserted into the center of the tumor and connectedto a pressure transducer. This makeup enables continuous and stablerecordings of fluid pressure. Fluid communication between the needle andthe transducer was confirmed by compression and decompression of thetubing during each measurement. Tumor IFP was measured once beforetreatment with PDGF aptamers, and again 1-2 hours after the lastadministration of aptamers or vehicle alone. The change in tumor IFP wascalculated for each tumor. After the second IFP measurement the ratswere sacrificed and the tumors were excised and snap frozen in liquidnitrogen for further analyses.

Example 3 Immunohistochemistry

For routine morphology, paraffin-embedded 4 μm sections were stainedwith van Gieson staining. Immunohistochemistry was performed on 6 μmcryosections from PROb tumors. Sections were fixed in acetone andblocked with 0.3% hydrogen peroxide in methanol for 15 minutes, rinsedand further incubated in a solution containing 20% human normal serum ina buffer containing 2% rat serum, 3% bovine serum albumin, 0.01% NP40 inPBS (RM buffer) for 5 hours at 4° C. Primary antibodies dissolved in RMbuffer were added, either 4 μg/mL affinity-purified rabbit anti-PDGFpreceptor IgG (Claesson-Welsh et al. (1988) Mol. Cell Biol. 8:3476-3486)for 5 hours at 4° C., or overnight at 4° C. with 1.3 μg/mL of themonoclonal mouse anti-PDGF-AB/BB IgG (PGF 007, Mochida PharmaceuticalCompany, Tokyo, Japan). Sections were rinsed in PBS with 0.01% NP40.Bound IgG was detected with biotinylated goat anti-rabbit orbiotinylated rabbit anti-mouse antibodies, respectively. Sections weredeveloped with a Vectastain ABC elite kit (Vector, Burlingame, Calif.)using amino-ethyl-carbazole as a chromophore. Sections werecounter-stained with Mayer's hematoxylin for 30 seconds.

Example 4 Treatment of KAT-4 Tumors with PDGF Inhibitors

SCID-mice bearing subcutaneous KAT-4 tumors (human, anaplastic thyroidcarcinoma) were pre-treated for 4 consecutive days with 12 mg×kg⁻¹×day⁻¹SELEX aptamers (i.p. injections, three times daily). Tumor interstitialfluid pressure was measured using the wick-in-needle technique. Theresults are set forth in FIG. 4. * p<0.05, Student's t-test.

Example 5 Treatment of KAT-4 Tumors with a PDGF Inhibitor in Combinationwith the Cytotoxic Agent Taxol

SCID-mice bearing subcutaneous KAT-4 tumors (human, anaplastic thyroidcarcinoma) were pre-treated for 4 consecutive days with 12 mg×kg⁻¹×day⁻¹SELEX aptamers (i.p. injections, three times daily). [³H]Taxol wasinjected s.c at a site distant from the tumor and in a mix of 5 mg×kg⁻¹unlabelled Taxol. Eight or 24 hours following injection of radiolabelleddrug, blood was sampled, animals were sacrificed and tumors and 4 othertissues were excised. Subsequently, tissues were weighed and homogenizedin a RIPA lysis buffer and the amount radioactivity in each sample wasdetermined in a scintillation counter. Tumor uptake of Taxol wasexpressed as cpm/g tumor tissue divided by cpm/ml blood. The results areset forth in FIG. 5. * p<0.05, Student's t-test.

Statistical analysis. Statistical analysis was performed using thepaired or unpaired two-sided Student's t-test. A p-value <0.05 wasconsidered statistically significant.

1. A therapeutic composition comprising a PDGF aptamer and a cytotoxicagent.
 2. The therapeutic composition of claim 1 wherein said PDGFaptamer is identified according to a method comprising: a) preparing acandidate mixture of nucleic acids; b) contacting the candidate mixtureof nucleic acids with PDGF, wherein nucleic acids having an increasedaffinity to PDGF relative to the candidate mixture may be partitionedfrom the remainder of the candidate mixture; c) partitioning theincreased affinity nucleic acids from the remainder of the candidatemixture; and d) amplifying the increased affinity nucleic acids to yielda mixture of nucleic acids enriched for nucleic acids with relativelyhigher affinity and specificity for binding to PDGF, whereby a nucleicacid ligand of PDGF may be identified.
 3. The therapeutic composition ofclaim 1 wherein said PDGF aptamer is:

wherein: U at positions 6, 20 and 30 is 2′-fluoro-2′-deoxyuridine. C atpositions 8, 21, 28, and 29 is 2′-fluoro-2′-deoxycytidine. G atpositions 9, 15, 17, and 31 is 2′-O-Methyl-2′-deoxyguanosine. A atposition 22 is 2′-O-Methyl-2′-deoxyadenosine; and


4. The therapeutic composition of claim 1 wherein said cytotoxic agentis selected from the group consisting of Bleomycin, Cisplatin, and Ptanalogues; Carboplatin and Iproplatin, Cyclophosphamide, Daunorubicin,Doxofluoridine, Doxorubicin, Etoposide, Epirubicin, 5-Flurouracil,Gemzar, Ifosfamide, Melphalan, Methotrexate, Mithramycin, Mitomycin C,Mitoxanthrone, Streptozotocin, Taxol and Taxotere, Vincristine,Vinblastine, Vindesine, Vinorelbine, Topotecan and CPT-11